The concept of multiple opioid receptors, first proposed almost 25 years ago, has become increasingly important. Evidence now suggests subtypes of both mu and kappa receptors. Pharmacological and binding approaches suggest that existence of mu1 and mu2 receptors. The mu2 site corresponds to the classical morphine-selective mu receptor identified in the guinea pig ileum bioassay. In addition to differences in their binding profiles, mu1 and mu2 actions are easily differentiated pharmacologically. Although both subtypes elicit analgesia, mu1 receptors act supraspinally while mu2 receptors act at the level of spinal cord. Furthermore, mu2 receptors mediate morphine's respiratory depression and inhibition of gastrointestinal transit. More recent work has suggested multiple kappa subtypes as well. The kappa1 subtype is defined by its selectivity for the agonists U50,488H and U69,593 and the antagonist nor-binaltorphimine. Studies from our laboratory with naloxone benzoylhydrazone (NalBzoH) identified a novel site (kappa3) with a binding profile unlike any previously described receptor. With a density in calf, rat and mouse brain 2-fold higher than either mu or delta receptors, kappa3 receptors are the predominant opioid receptor in the CNS. Pharmacologically, kappa3 receptors elicit analgesia through a supraspinal mechanism which is easily separated from mu, delta and kappa1 receptor mechanisms. Additional studies now suggest that kappa3 receptors correspond to the "N", or nalorphine, receptor first proposed by Martin in his concept of "Receptor Dualism". A human neuroblastoma cell line expressing functionally active mu and kappa3 receptors has now been identified. Morphine (IC50 0.2 muM) and NalBzoH (IC50 2 muM) both inhibited forskolin-stimulated cyclase by 80% through discrete mu and kappa3 receptor mechanisms, respectively. In the present application, we propose to continue our studies of mu1, mu2 and kappa3 subtypes in this cell line. Studies will characterize the binding of the subtypes and their actions in function assays, specifically forskolin-stimulated cyclase. Experiments will investigate the effects of chronic agonist and antagonist exposure on function and receptor turnover. Our second aim is to utilize the selective mu1, mu2 and kappa3 assays developed in our laboratory to examine opioid binding in brain. Binding levels and selectivities will be examined in various species. The developmental appearance of kappa3 site will be compared to the other subtypes and its regional distributions will be examined autoradiographically. Finally, we will extend our studies utilizing affinity labels synthesized in our laboratory to investigate binding heterogeneity at the molecular level. Through an understanding of opioid receptor multiplicity we hope to better use the analgesics currently available and perhaps to develop new approaches to the design of novel, innovative drugs lacking the problematic side-effects of morphine and its analogs.